The mitochondrial DNA common deletion as a potential biomarker of cancer-associated fibroblasts from skin basal and squamous cell carcinomas

Cancer-associated fibroblasts (CAFs) are components of the tumor microenvironment and represent appealing therapeutic targets for translational studies. Conventional protein-based biomarkers for CAFs have been reported to be limited in their specificity, rendering difficult the identification of CAFs from normal fibroblasts (NFs) in clinical samples and dampening the development of CAF-targeted therapies to treat cancer. In this study, we propose the mitochondrial RNA and the mitochondrial DNA (mtDNA) common deletion (CD) as novel indicators of CAF identity. We found that cancer-activation correlated with decreased levels of the mtDNA CD, a condition not due to altered mitochondria count or cellular redox state, but potentially linked to the generalized overexpression of mtDNA maintenance genes in CAFs. Decreased mtDNA CD content in CAFs was associated with moderate to strong overexpression of mtDNA-encoded genes and to slightly improved mitochondrial function. We identified similar patterns of upregulation of mtDNA-encoded genes in independent single-cell RNA seq data obtained from squamous cell carcinoma (SCC) patients. By using the identified nucleic acids-based indicators, identification of CAFs from NFs could be improved, leading to potential therapeutic benefits in advancing translational and clinical studies.

Figure 1.BCC and SCC CAFs harbor decreased levels of the mtDNA CD as compared to NFs. (A) Evaluating expression of conventional CAF biomarkers in BCC CAFs (left) and SCC CAFs (right) by qPCR.The expression levels of the indicated genes were expressed as fold change as compared to NFs. n = 3 biological replicates, statistical analyses: unpaired Wilcoxon t test for BCC CAFs, paired Mann-Whitney t test for SCC CAFs.(B) WBs for the expression of α-SMA in NFs and SCC CAFs from the same donors.Left: representative WBs from donor-matched NFs and SCC CAFs at low passages (P2 and P4).Right: quantitation of α-SMA levels from n = 15 NFs and SCC CAFs pairs.The expression of α-SMA in SCC CAFs is expressed as fold change as compared to NFs, variability is depicted as SD, statistical analysis: paired t test.(C) Human mtDNA, containing genes encoding proteins of the electron transport chain (ETC), Humanin, rRNAs and tRNAs.The ~ 5 kb region affected by the common deletion (CD) is flanked by two 13 bp direct repeats.(D) Schematic representations of WT and CD mtDNA molecules showing localization of the primers for the analysis of total., WT and CD mtDNA copies within the mitochondrial genome.(E) qPCR for the relative quantification of mtDNA WT and CD in NFs, BCC CAFs and SCC CAFs.n = 6 biological replicates.(F) Citrate synthase (CS) activity in NFs, BCC CAFs and SCC CAFs.The CS activity in BCC and SCC CAFs is expressed as fold change as compared to NFs. n = 3 biological replicates.(G) Left: representative microscopic images of NFs, BCC and SCC CAFs stained with Mitotracker Red for membrane polarization and DAPI for nuclei identification.Right: quantification of total mitochondrial area normalized for cell count.AU arbitrary units.n = 3 biological replicates.(H) Measurement of ROS in NFs, BCC CAFs and SCC CAFs by DCF assay.n = 3 biological replicates.For panels (E-H) statistical analysis was Kruskal-Wallis test with Dunn's post hoc test comparing to NFs.To further corroborate this observation, we imaged NFs and CAFs stained with MitoTracker Red, a fluorescent dye that detects intact and functional mitochondria, and DAPI, providing nuclear staining for parallel cell count (Fig. 1G).Microscopic analysis did not reveal significant differences in the amounts of mitochondria per cell, as measured by the ratio between the cumulative area of the MitoTracker Red staining and the total amount of cells in the monitored fields.Variations in the levels of the mtDNA CD have been correlated with oxidative stress and the mutagenic effects of reactive oxygen species (ROS) on the mtDNA 23 ; thus, we also monitored the basal levels of total cellular ROS in NFs and CAFs using the established 2′,7′-Dichlorofluorescin diacetate (DCF) fluorescent reporter assay.We did not detect alterations in the basal ROS content in NFs and CAFs (Fig. 1H).

Several mtDNA maintenance genes are upregulated in primary BCC and SCC CAFs
We measured by qPCR the transcriptional levels of a panel of candidate genes encoding proteins involved in mtDNA replication, repair and degradation; all mechanisms whose dysfunctions have been linked to mtDNA CD formation and maintenance 14 .We found that the majority of the interrogated genes show an upregulation trend in both BCC CAFs (Fig. 2A) and SCC CAFs (Fig. 2B) as compared to NFs.In particular, the expression of TFAM, encoding an mtDNA-binding protein organizing nucleoids and playing crucial roles in the maintenance of the mitochondrial genome, is upregulated in both CAF population (average fold change of ~ 4. To test whether transcriptional upregulation would lead to a concomitant increase in protein levels, we assessed the levels of PARP1 and TFAM, two factors that play key roles in mtDNA maintenance 24,25 in NFs and CAFs (Fig. 2C).In good agreement with the qPCR data, we found upregulation of the protein levels of PARP1 (average fold change of ~ 2.3 in BCC and SCC CAFs) and TFAM (average fold change of ~ 2.4 in BCC CAFs and ~ 2.6 in SCC CAFs) in both CAF populations.

mtDNA-encoded gene expression is altered in primary BCC and SCC CAFs, leading to moderately improved mitochondrial metabolism
Having detected a decrease in the mtDNA CD content and a concomitant increase in the mtDNA WT proportion in BCC and SCC CAFs as compared to NFs, we sought to determine the expression levels of mtDNA-encoded genes and the functionality of the electron transport chain (ETC) machinery.We initially tested by qPCR the transcriptional levels of mtDNA-encoded genes and found different trends between two CAF types (Fig. 3A).In BCC CAFs, most mtDNA-encoded genes did not show altered expression, with the exceptions of the MT-RNR1 and MT-RNR2 genes, encoding the mitochondrial-specific 12s and 16s ribosomal RNA subunits, and the Humanin open reading frame, encoding a micropeptide involved in stress responses and inflammation, which show an average fold change of ~ 6.0, 3.1 and ~ 9.3 as compared to NFs, respectively.In SCC CAFs, all the mtDNA-encoded genes showed a marked upregulation as compared to NFs, with average fold changes ranging from ~ 15 of MT-ND6 and ~ 380 of MT-RNR1.
It has been shown that the levels of polycistronic RNA transcribed from mtDNA molecules do not strictly correlate with increased translation of the corresponding encoded proteins 26 .To test whether the transcriptional levels of the mtDNA-encoded genes assessed by qPCR translates into similar patterns of protein expression, we monitored by WB the levels of MTCO2, encoded by the MT-CO2 mtDNA gene, in NFs and CAFs (Fig. 3B).In agreement with the transcriptional data, we found that MTCO2 expression is unaltered in BCC CAFs and upregulated in SCC CAFs as compared to NFs.
Having detected differences in the expression patterns of mtDNA-encoded genes in the two populations of CAFs as compared to NFs, we then sought to perform functional assays to determine the activity of the ETC and the overall rates of mitochondrial metabolism in NFs and CAFs.Using the established Seahorse assay we simultaneously measured the oxygen consumption rates (OCR) and extracellular acidification rate (ECAR), monitoring mitochondrial respiration and glycolysis in live cells, respectively.The OCR was determined in both basal conditions, i.e. before addition of chemical modulators of the activity of the ETC, and maximal conditions, i.e. after addition of the protonophore FCCP to collapse the inner mitochondrial membrane gradient (Fig. 3C).We found that both BCC and SCC CAFs display slightly increased OCR as compared to NFs in basal state and more marked increased OCR in maximal conditions, albeit with high variability among biological replicates.Comparing patient-matched NFs and SCC CAFs pairs showed a significant effect of cancer status (Fig. 3D).We also monitored the ECAR, which arises from acidification of the culture medium due to lactate diffusion in the culture medium as a proxy for glycolytic activity.The ECAR was monitored in both basal conditions, i.e. before addition of chemical modulators of activity of the ETC, and maximal conditions, i.e. after addition of FCCP (Fig. 3E).ECAR quantifications revealed a significant effect of cell type (2-way ANOVA P = 0.006) but no significant differences in basal rate upon post-hoc testing.Upon FCCP treatment BCCs had a significantly higher rate compared to NF and SCC (Fig. 3E).As an orthogonal measure of mitochondrial metabolism, we monitored  the mitochondrial membrane potential by quantifying the intensity of the internalized MitoTacker Red staining normalized on a per cell basis (Fig. 3F).While both CAF populations tended have higher membrane potential, only BCC was significantly higher.

Single-cell data from SCC CAFs reveals upregulation of mtDNA-encoded genes
We analysed single-cell RNA sequencing data from a previous study 27 that compared patient-matched NFs and SCC CAFs.To separate the fibroblasts pool among the mixed cell population contained in the datasets we performed clustering and UMAP projection, clustering sequenced cells based on expression of single and/or  combinations of gene markers (Fig. 4A).This analysis retrieved eleven cellular clusters of similar composition to the original publication.We observed that in samples from healthy skin regions and patient-matched cancerproximal tissues the fibroblasts pool was limited, representing ~ 5% or less of total cells.Among the twelve SCC patients from which cells where sampled, we identified six patients where the population of sequenced fibroblasts was sufficiently represented, allowing subsequent analyses.We profiled the cumulative expression of twelve conventional CAF biomarkers and mtDNA-encoded genes in the six patients (Fig. 4B).This analysis revealed that the expression of the selected conventional CAF biomarkers was highly variable and did not consistently differ between CAFs and matched NFs.Conversely, we detected a more consistent upregulation trend in the cumulative expression of mtDNA-encoded genes in CAFs of the six patients.
To evaluate potential correlations between the expression of the genes of interest and cancer-activation, we monitored the expression of conventional CAF biomarkers and mtDNA-encoded genes in a cumulative fashion within the cell clusters identified by the UMAP analysis or as single entities within the fibroblasts cluster and within samples derived from NFs and SCC CAFs.The cumulative analysis of conventional CAF biomarkers in the eleven cell clusters derived from the UMAP analysis revealed that these genes do not display marked upregulation trends in any clusters (Fig. 4C).When individual genes were in the fibroblast cluster, CXCL1, FAP and PDGFRA displayed moderately higher levels in SCC CAFs as compared to NFs with high variability (Fig. 4D).We repeated these analyses for the mtDNA-encoded genes.By monitoring their cumulative expression in the eleven UMAP-identified clusters, we found that in the skin, immune and endothelial cells clusters the mtDNAencoded genes were upregulated; interestingly, the highest degree of upregulation was observed in the fibroblasts cluster (Fig. 4E).The analysis of mtDNA-encoded genes within the fibroblast cluster revealed that multiple genes, especially MT-CO1, MT-CO2 and MT-ND5, were upregulated in SCC CAFs of all patients (Fig. 4F).

Discussion
The CD as a biomarker for tumorigenesis has been studied 15 ; in some cancers, the CD is highly abundant, while in others it is not detected.Accumulation of the CD has been consistently linked to the emergence of multisystemic mitochondrial pathologies, such as the Kearns-Sayre syndrome (KSS) 28,29 and progressive external ophthalmoplegia (PEO) 29 .KSS and PEO encephalomyopathies manifest dysfunctions in the nervous, muscular and endocrine system.On a molecular level, cells derived from KSS and/or PEO patients show elevated levels of CD heteroplasmy, accompanied by dysfunctional mtDNA gene expression, impaired oxidative phosphorylation and a decrease in cellular energy production 30,31 .Potential correlations between variable CD levels in CAFs were not so far reported.We find that in SCC and BCC CAFs, the CD levels are significantly lower in comparison to NFs (Fig. 1E), suggesting that the CD is a potential molecular biomarker to discriminate CAFs from NFs.To our knowledge this observation has not been previously reported.Previous studies link increased CD levels to aging 16,17 ; therefore, the observed elevated levels of the CD in NFs as compared to SCC and BCC CAFs could be explained by the fact that our NFs and corresponding CAFs derive from aged patients.While the molecular mechanism of CD formation and maintenance is not well understood, it has been reported that ROS is associated with its formation 23 .However, we did not detect alterations in the ROS basal content in NFs and CAFs (Fig. 1H), suggesting that oxidative stress may not play a key role in determining the diverse heteroplasmic contents of the CD between NFs and CAFs.
Seeking potential explanations for the decreased levels of the mtDNA CD in BCC and SCC CAFs and compared to NFs, we hypothesized that cancer activation could reprogram the expression patterns of genes encoding mtDNA maintenance proteins.Following this rationale, CAFs would prevent the formation of the CD or degrade mtDNA molecules which acquire the CD.To investigate this hypothesis, we measured by qPCR the transcriptional levels of a panel of candidate genes encoding proteins involved in mtDNA replication, repair and degradation (Fig. 2A,B), all mechanisms whose dysfunctions have been linked to mtDNA CD formation 14,32 .We found that the majority of the interrogated genes showed an upregulation trend in both SCC and BCC CAFs compared to NFs.These data suggest that the expression of genes encoding proteins involved in mtDNA maintenance (i.e.replication, degradation and BER), all pathways whose dysfunctions have been linked to the emergence of mtDNA deletions and the mtDNA CD, is upregulated in CAFs.This observation could be explained by hyperactive mtDNA maintenance pathways in CAFs, thereby resulting in improved clearance and/or reduced formation of the CD in CAFs as compared to NFs.
Mitochondrial-encoded genes have not yet been reported as biomarkers for CAFs.We observed a general increase in transcriptional levels of mtDNA-encoded genes for BCC and SCC CAFs (Fig. 3A,B, respectively).Surprising to us, the SCC CAFs had very significant overexpression as compared to NFs (e.g.~ 380-fold increase of MT-RNR1), much more so than for the BCC CAFs.This observation could be explained by the fact that SCC often is associated with a stronger cancer phenotype in comparison to BCC.For example, BCC cells tend to grow less invasively and are attributed to less severe clinical consequences than SCC 33,34 .In our functional assays for mitochondrial metabolism, we observed a moderate increase in the OCR and ECAR in both SCC and BCC CAFs compared to NFs (Fig. 3C-E).Tumor growth is associated with altered mitochondrial metabolism and the modulation of oxygen consumption is reported as a rate limiting substrate for tumorigenesis 35 .In skin cancer, as well as most cancer types, hypoxic conditions in precancerous tissue promote cancer formation 36 .This might explain the observation for increased OCR in our SCC and BCCs, such that as cancer develops and tumor mass increases, oxygen consumption will increase due to the rise in actively proliferating cells 37 .Taken together, these findings suggest that the elevated heteroplasmic content of wild-type mtDNA molecules in CAFs as compared to NFs lead to increased transcription of mtDNA-encoded genes, with significant differences of expression rates among BCC and SCC CAFs.This translates in moderately increased mitochondrial activity, suggesting that the suppression of the deleted mtDNA population observed in CAFs may modestly affect mitochondrial metabolism.
Finally, we were pleased to observe that in an independent study that previously reported single-cell RNA sequencing data in SCC patients 27 , a corroboration to our qPCR data showing the upregulation of mitochondrial-encoded genes in SCC CAFs (Fig. 3A).The fact that we could observe an overall upregulation for most mitochondrial genes in the fibroblast cluster, suggests that this phenotype may be ascribed to SCC CAFs down to the single cell level.
In summary, our findings show that primary BCC-and SCC-derived CAFs display significantly reduced contents of the mtDNA CD as compared to isogenic NFs.In both SCC and BCC CAFs, we consistently observed a pattern of upregulation in genes involved in mtDNA maintenance; thus, molecular mechanisms selectively removing and/or preventing the formation the mtDNA CD could be hyperactive in CAFs.In addition, SCC and BCC CAFs with increased WT mtDNA levels displayed enhanced expression of mtDNA-encoded genes and moderately improved mitochondrial functionality.We validated the increased expression of the mtDNAencoded genes by evaluating independent single-cell RNA sequencing data from SCC CAFs, which corroborated our qPCR data.In conclusion, we propose that the mtDNA CD heteroplasmic content and the expression of mtDNA-encoded genes represent the potential for robust and specific biomarkers to discriminate CAFs from NFs.These suspected biomarkers may hold promise to advance clinical and translational studies aimed to understand CAF function and biology.

Patients and clinical samples
Fibroblasts from basal cell carcinoma (BCC) and normal fibroblasts (NFs) from healthy skin were obtained from the Biobank of the dermatology department of the University Hospital Zurich managed by SINTEGRITY.CH research program.Fibroblasts from squamous cell carcinoma (SCC) and NFs from healthy skin were obtained from Beer laboratory at the University Hospital Zurich.SCCs and surplus human biopsies used to isolate cells were collected after obtaining patient informed written consent and upon approval from local ethical committees and were conducted according to the principles of the Declaration of Helsinki.The information relative to patients are reported in Table S1.

Primary fibroblasts cultures
Normal fibroblasts (NFs) and CAFs were isolated from the skin of healthy and BCC or SCC patients using the procedure reported in [38][39][40][41] .Isolated cells were then cultured in Dulbecco's modified Eagle medium (DMEM), high glucose, pyruvate (ThermoFisher Scientific, USA) with 10% fetal bovine serum (FBS, ThermoFisher Scientific, USA).For BCC CAFs and associated NFs the medium was supplemented with 100 μg/ml Normocin (Invivogen, USA).For SCC and associated NFs the medium was supplemented with 100 units/ml of Penicillin and 100 µg/ ml of Streptomycin (ThermoFisher Scientific, USA).Cells were routinely passaged every 2-3 days in 10 cm Petri dishes and/or T75 flasks and detached with a solution of Trypsin-EDTA 0.25% and phenol red (ThermoFisher Scientific, USA).Experiments were performed at passages comprised between 2 and 6.

RNA and mtDNA extraction
Cells were grown to ~ 70-80% confluency, harvested by scraping and pelleted by centrifuging.RNA was isolated from cells pellets from NFs, BCC and SCC CAFs using the RNeasy Mini Kit (Qiagen, Germany) following manufacturer's instructions.The RNA preparation included an on-column RNase-Free DNase (Qiagen, Germany) treatment prior to elution.mtDNA was isolated from cells pellets using the protocol described in 42 with minor changes.Briefly, cell pellets were resuspended in 250 µl Buffer P1 from the QIAprep Spin Miniprep Kit (Qiagen, Germany) supplemented with RNAse A. The resuspended cells were sonicated using a Vibra-Cell Ultrasonic Liquid Processor (Sonics and Material, model VCX750, USA) with three pulses of 30 s each, at maximum amplitude, with intervals of 2 min in ice.The mtDNA was isolated following the QIAprep Spin Miniprep Kit instructions, and further purified using the Agencourt AMPure XP (Beckman Coulter, USA) paramagnetic beads.Concentrations of purified RNA and mtDNA was assessed using a NanoDrop (ThermoFisher Scientific, USA) spectrophotometer.

Quantitative real-time PCR (qPCR) for the analysis of gene expression levels
1-2 µg of purified RNA were retrotranscribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, USA) following manufacturer's instructions.Each qPCR sample consisted in 4-6 ng of cDNA, 5 × HOT FIREPol EvaGreen qPCR Mix (Solis Biodyne, Estonia), 0.4 μM forward primer, 0.4 μM reverse primer and nuclease-free water to a total of 12 µl.The primers used in this study are listed in Table S2 and were obtained from Eurogentec, Belgium.qPCRs were conducted using a Rotor-Gene 6000 (Corbett Research, Australia) device, using the following protocol for each cycle: 95 °C for 30 s, 60 °C for 1 min, 40-50 cycles, with fluorescence acquisition at the end of the last amplification step.The expression levels for nuclear genome-encoded target genes were assessed in each biological condition using the delta-delta Ct method using the expression of GAPDH or ACTB housekeeping genes as normalizers.The expression levels for mtDNA-encoded target genes were assessed in each biological condition using the delta-delta Ct method using the expression of an untranslated region of the polycistronic mtDNA-encoded RNAs as normalizer.Each combination of cDNA and primers was assayed in three technical replicates.The number of biological replicates relative to each experiment are indicated in the Figure legends.

Quantitative real-time PCR (qPCR) for the analysis of mtDNA species
5-10 ng of purified and RNA-free mtDNA was assayed by qPCR using the GoTaq 2 × qPCR Master Mix (Promega, USA), 0.4 μM forward primer, 0.4 μM reverse primer and nuclease-free water to a total of 12 µl per sample.The primers used in this study are listed in Table S2 and were obtained from Eurogentec, Belgium.qPCRs were conducted using the Rotor-Gene 6000 (Corbett Research, Australia) device, following a standard qPCR protocol.Normalization was performed with the delta-delta Ct method using the qPCR data for the amplification of the total mtDNA content in each sample as normalizer.The corresponding values obtained from the qPCR for the mtDNA CD and undeleted species were expressed as relative fractions over the corresponding total mtDNA.The levels of genomic DNA contamination in the mtDNA preparation were routinely assessed by performing parallel qPCRs on a genomic region of the ACTB gene, resulting in low or absent signal.Each combination of mtDNA and primers was assayed in three technical replicates.The number of biological replicates relative to each experiment are indicated in the figure legends.

Western blotting
NFs, BCC and SCC CAFs were grown to confluency of ~ 80%, and procedures for western blotting followed two protocols.For the western blotting depicted in Fig. 1B, cells were collected by scraping in SDS loading buffer composed as follows: 50 mM Tris-HCl pH 6.8, 2% SDS, 10% Glycerol, 0.025% Bromophenol Blue, supplemented with freshly added 100 mM DTT (all reagents from Sigma-Aldrich, USA).Cells were resuspended, boiled and sonicated.Proteins were separated on SDS-PAGE gels with Mini-PROTEAN Tetra vertical electrophoresis cell (Bio-Rad Laboratories Inc., USA) alongside a Prestained Protein MW Marker (ThermoScientific, USA) and transferred to an Amersham Protran 0.2 µm nitrocellulose membrane (GE Healthcare, USA).The membranes were blocked for 1 h at room temperature with 3% non-fat dried milk in PBS supplemented with 0.5% Tween20 (Sigma-Aldrich, USA) and incubated overnight at 4 °C with primary antibodies diluted in PBS-0.5% Tween20 supplemented with 3% non-fat dried milk.After extensive washes in PBS-0.5% Tween20, secondary antibodies were incubated for 1 h at room temperature.Proteins were detected using BCIP/NBT Color Development Substrate (Promega, USA) and membranes were scanned using a LiDE210 scanner (Canon, Japan).Band intensity was quantified using ImageJ software (NIH, USA) and normalized to β-Actin levels.A second western blot was performed with normalized protein amounts and used for subsequent quantifications and assembly of the panels.
For the western blots depicted in Figs.2C and 3B, cells were harvested by scraping and cell pellets were washed with cold PBS.Whole cell lysates were obtained by lysis with RIPA Lysis and Extraction Buffer (ThermoFisher, USA) supplemented with Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche, Switzerland).Protein concentration was assessed with the Pierce BCA protein assay kit (ThermoFisher, USA) and 20 µg of WCE were loaded on a NuPAGE 4-12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well (Invitrogen, USA), alongside the PageRuler Prestained Protein Ladder (ThermoFisher, USA).Electrophoretic run was performed using the MES SDS Running Buffer (ThermoFisher, USA).Proteins were blotted on the Trans-Blot Turbo mini-size PVDF membrane (Bio-Rad Laboratories Inc., USA).The membrane was blocked for 1 h with 5% non-fat dry milk diluted in Tris-buffered Saline Buffer supplemented with 0.1% Tween20 (TBS-T).After extensive washing in TBS-T, membranes were incubated with primary antibodies diluted in TBST-T with 5% dry milk and incubated overnight at 4 °C.After extensive washing with TBS-T, incubation with secondary antibodies diluted in TBST-T with 5% dry milk for 1 h at room temperature.After washing, the membrane was incubated with the ECL Western Blotting Substrate (Promega, USA) and signals were acquired with a ChemiDoc MP imaging system (Bio-Rad Laboratories Inc., USA).Quantification of bands was performed using the Fiji software 43 .The number of quantified biological replicates are indicated in the figure legends.The primary and secondary antibodies used in this study are listed in the Key resources table (Table 1).

Citrate synthase (CS) assay
1.5 × 10 4 NFs, BCC and SCC CAFs were seeded in 96 well-plates and assayed when a confluency of ~ 70-80% was reached.The activity of the citrate synthase enzyme, a proxy for cellular mitochondria content, was measured using the Citrate Synthase Activity Assay Kit (Abcam, UK) following manufacturer's instructions.The colorimetric readings were acquired using a microplate Tecan instrument (Tecan, Infinite 200 PRO model, Switzerland) and normalized to a cell-free blank and to the amounts of harvested cells for each sample.Each condition was assayed in three biological replicates, and each sample was tested in three technical replicates.

Measurement of reactive oxygen species (ROS)
1.5 × 10 4 NFs, BCC and SCC CAFs were seeded in 96 well-plates and assayed at a confluency of ~ 70-80%.Culture medium was removed and replaced with 200 µl per well of a 50 µM 2′,7′-Dichlorofluorescin diacetate (DCF, Sigma Aldrich, USA) solution in PBS 1 ×.Plates were incubated for 30 min in the incubator to allow internalization of the chemical probe.The solution was removed, and 30 µl trypsin solution were added.Following 3-5 min incubation where detachment of the cells occurred, 120 µl PBS 1 × were added and fluorescence was acquired using a CytoFLEX flow cytometer (Beckman Coulter, USA).Data were normalized internally by gating.Cell-free blank controls and unstained controls treated in parallel but where DCF was omitted were as well performed and used as control for normalization.Each condition was assayed in three biological replicates, and each sample was tested in three technical replicates.

Seahorse assays
Cellular oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) were measured using the Seahorse Cell Metabolism Analyzer XF96.Cells from each patient-derived line were plated in 8 replicate wells at a density of 6000 cells/well in a 96-well Seahorse plate.After 24 h, media was changed to unbuffered XF assay media with 11 mmol/l glucose, 2 mmol/l glutamine and pyruvate at pH 7.4.Following basal measurement, oligomycin, FCCP, and rotenone/antimycin A were sequentially injected to achieve final concentrations of 1, 1.5, and

Imaging
Cells from each patient-derived line were plated in three replicate wells at a density of 3000 cells/well in a 96 well plate.After 24 h, cells were stained with 200 nM Mito Tracker Deep Red FM (ThermoFisher Scientific) and 200 ng/ml Hoechst in phenol-free DMEM with no serum for 20 min.Staining media was then replaced with phenol-free DMEM with 10% FBS and cells were imaged with a BioTek Cytation 5 using a 10 × objective.Mitochondrial area was segmented and quantified, and average Mito Tracker Red intensity was quantified within mitochondrial area using BioTek Gen5 (version 3.11).

Analysis of single-cell sequencing data
Single cell RNA seq data on patient-matched normal and squamous cell carcinoma samples were obtained from GSE144240 27 .BAM files were downloaded from the NIH Sequence Resource Archive and converted to fastq files using the bamtofastq function in cellranger (version 3.1.0).Fastq files were then analyzed using the count function from cellranger.The GRCh38-2020-a reference from cellranger was used.Seurat 44 https://doi.org/10.1038/s41598-023-50213-1www.nature.com/scientificreports/ 2 in BCC CAFs and ~ 16.6 in SCC CAFs).Similarly, genes encoding regulators of mtDNA replication, including MGME1 (average fold change of ~ 2.7 in BCC CAFs and ~ 10.6 in SCC CAFs), TWNK (average fold change of ~ 1.9 in BCC CAFs and ~ 9.5 in SCC CAFs), POLG (average fold change of ~ 2.7 in BCC CAFs and ~ 11 in SCC CAFs), POLG2 (average fold change of ~ 3.6 in BCC CAFs and ~ 10.8 in SCC CAFs), TEFM (average fold change of ~ 2.2 in BCC CAFs and ~ 9.9 in SCC CAFs) TFB2M (average fold change of ~ 3.5 in BCC CAFs and ~ 9.7 in SCC CAFs) and TOP1MT (average fold change of ~ 1.7 in BCC CAFs and ~ 9.2 in SCC CAFs) are consistently upregulated in both CAF populations.The expression of genes encoding enzymes participating in the base excision repair (BER) pathway (see Fig.2A,Bfor associated BER genes, indicated in blue circles), the only mtDNA repair mechanism proven to occur in mitochondria in vivo, also displayed an upregulation in both CAF populations.The expression of proteins involved in the mismatch repair and double-strand break repair pathways, two repair mechanisms with little in vivo evidence to play a role in mitochondria, display diverse patterns of up-and down-regulation between BCC and SCC CAFs. https://doi.org/10.1038/s41598-023-50213-1

Figure 2 .
Figure 2. Expression of key genes involved in mtDNA maintenance is upregulated in BCC and SCC CAFs.qPCR analysis for the expression of nuclear DNA-encoded genes involved in mtDNA maintenance processes, i.e. mtDNA degradation, replication and repair (see legends at the bottom of panels).Experiments were performed in BCC CAFs (the asterisk indicates a comparison surviving Benjamini-Hochberg False Discovery Rate correction) (A) and SCC CAFs (B), and changes in expression levels were expressed as fold changes on a log scale over matching NFs.n = 3 biological replicates were used.Statistical analysis was performed with an unpaired Wilcoxon t test for BCC CAFs and with a paired Mann-Whitney t test for SCC CAFs.(C) WBs were performed to monitor the expression levels of PARP1 and TFAM protein in NFs, BCC and SCC CAFs.Left: representative WBs panels.Right: quantitation of WBs for PARP1 and TFAM.Expression levels in BCC and SCC CAFs are expressed as fold change as compared to NFs. n = 3 biological replicates were used.Statistical analysis for BCC CAFs was conducted with a Mann Whitney unpaired t test and for SCC CAFs with a paired Wilcoxon t test.

Figure 3 .
Figure 3. Expression of mtDNA protein-coding genes is upregulated in BCC and SCC CAFs.(A) qPCR analysis for the expression of mtDNA-encoded genes in BCC CAFs (upper panel) and SCC CAFs (bottom panel), expressed as fold change over isogenic NFs (red dotted line).Color codes indicate the ETC complexes to which the encoded proteins belong to, rRNAs and the Humanin micropeptide.n = 3 biological replicates were used for each graph.Statistical analysis was performed with an unpaired Wilcoxon t test for BCC CAFs and with a paired Mann-Whitney t test for SCC CAFs (the asterisk indicates a comparison surviving Benjamini-Hochberg False Discovery Rate correction).(B) WB analysis for the protein levels of MTCO2 in BCC and SCC CAFs.Upper panel: representative WBs.Arrowhead indicates MTCO2, asterisk designates a cross-reactive unspecific band.Bottom panel: quantitation of MTCO2 expression in n = 3 biological replicates, expressed as fold change over NFs.Statistical analysis was performed with a Wilcoxon t test.(C) Oxygen consumption rates (OCR) at basal and stimulated (maximal) ETC activity in NFs, BCC and SCC CAFs.n = 3 biological replicates were used and analyzed by Kruskal-Wallis test with Dunn's post hoc test.(D) OCR in basal and maximal conditions in three patient-matched NFs and SCC CAFs pairs analyzed by repeated measures two-way ANOVA with Sidak post-hoc test.(E) Extracellular acidification rates (ECAR) at basal and maximal ETC activity in NFs, BCC and SCC CAFs.Results relative to n = 3 biological replicates and analyzed by repeated measures 2-way ANOVA with Sidak post-hoc test.(F) Quantification of the intensity of Mitotracker Red staining normalized for cell count.Data are expressed as fold change over NFs and derive from n = 3 biological replicates.Analyzed by Kruskal-Wallis test with Dunn's post hoc test.

Figure 4 .
Figure 4. Single cell sequencing data corroborates the upregulation of mtDNA-encoded genes in SCC CAFs.(A) UMAP analysis of single-cell sequencing data collected by Ji et al. 27 from primary SCC CAFs and matched NFs from twelve donors.Eleven clusters were identified based on the expression of the gene markers listed in the legend.The fibroblasts cluster, accounting for ~ 5% of the sequenced cells, was analyzed further.(B) Analysis of the expression of conventional CAF biomarkers (grey bars, genes indicated in the legend) and mtDNA-encoded genes (green bars, genes indicated in the legend) in NFs and SCC CAFs from six patients with sufficient fibroblasts sequenced.NFs were set to 1 and relative expression fold changes in Log2 scale were calculated for each NFs and CAF pair (the asterisk indicates a comparison surviving Benjamini-Hochberg False Discovery Rate correction).(C) Relative expression of conventional CAF biomarkers in the eleven clusters identified by the UMAP analysis.(D) Relative expression of twelve conventional CAF biomarkers analyzed in the UMAP cluster 9, i.e. the fibroblasts cluster.(E) Relative expression of mtDNA-encoded genes in the eleven clusters identified by the UMAP analysis.(F) Relative expression of mtDNA encoded genes analyzed in the UMAP cluster 9, i.e. the fibroblasts cluster. https://doi.org/10.1038/s41598-023-50213-1www.nature.com/scientificreports/

Table 1 .
objects were created using the outputs from cellranger.Normal and cancer cell data from the same patients were combined into a single Seurat object (Seurat version 2.3.4) in R (version 4.1.2).Cells with fewer than 200 RNA features and greater than 25% of reads assigned to mitochondrial DNA were filtered.Clusters were assigned a fibroblast Key resource table.